Establishment and evaluation of a stable CHO cell line in which the nanobody PD-L1-Fc gene is precisely targeted into the C12orf35 locus

Abstract

Chinese hamster ovary (CHO) cells are an extensively used platform for manufacturing biopharmaceuticals, and nearly 80% of recombinant protein is produced by CHO cell lines. Randomly incorporating genes of interest into the genome is a common method for the development of stable CHO cell lines in industry, but it is vulnerable to genetic instability, is difficult to predict productivity, and is accompanied by a time-consuming and laborious screening process. Nonetheless, highly productive clones isolated from a randomized pool often exhibit unfavorable properties including transgene copy number loss and epigenetic silencing over the lifespan of the culture, ultimately lowering transgene transcription and corresponding recombinant protein production, which referred to as production instability. Thus, this challenging situation underscores the urgent desire for a new strategy to satisfy ever-growing industrial production requirements. The lack of specificity of gene integration, which is often susceptible to genetic instability, causes production instability. Alternatively, in recent years, many investigators have shown that the bottleneck arising from traditional randomized cell line development can be overcome through site-specific integration to insert exogenous pieces of DNA into a precise location, which permits their predictable function, allows high levels of transgene expression and makes it possible to generate homogeneous clones with consistent productivity and stability. The transcription and expression activities of genes are influenced by chromatin structural properties and the environment surrounding the genome, and loci that are capable of facilitating high and stable transgene transcription and expression are termed 'hotspots'. Many significant upfront advances have been made to identify potential hotspots, and a number of promising genome loci have been reported, such as the Hprt, Ywhae, Hipp11, Rosa26, and C12orf35 loci. The C12orf35 gene is located on a telomeric region of chromosome 8 in CHO cells. It is widely known that telomeres are usually noncoding, repetitive sequences distributed at chromosome terminals that act as buffers for those coding sequences further behind and thus enable foreign gene expression without interrupting functional genes. Studies have demonstrated that the C12orf35 gene is a potential locus for the integration of foreign genes in mammalian cells and that disruption of C12orf35 gene expression leads to increased productivities and shorter recovery times during selection pressure in CHO cells. Although the C12orf35 gene has been partially researched in cell line development, very few publicly available reports have systematically validated site-specific integration in cell lines concerning the stability of transgene passage, transgene transcription and expression levels. A range of studies have successfully utilized site-specific recombinase or genome editing tools to incorporate exogenous genes into the desired site in the CHO genome. Clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR-Cas9), a leading gene editing tool, uses a guide RNA to target the DNA sequence with the Cas9 enzyme to induce cuts and allows easy, efficient and cost-effective edting. CRISPR-Cas9 has already been applied to mediate the insertion of targeted genes in mammalian cells, including CHO cells, for fundamental research. However, the adoption of this technology for industrial purposes remains to be investigated. Therefore, in this study, we sought to establish a CRISPR/Cas9-mediated site-specific integration strategy to overcome existing weaknesses and lay the foundation for the development of industrial rCHO cell lines.

1. Introduction

Chinese hamster ovary (CHO) cells are an extensively used platform for manufacturing biopharmaceuticals, and nearly 80% of recombinant protein is produced by CHO cell lines. Randomly incorporating genes of interest into the genome is a common method for the development of stable CHO cell lines in industry, but it is vulnerable to genetic instability, is difficult to predict productivity, and is accompanied by a time-consuming and laborious screening process. Nonetheless, highly productive clones isolated from a randomized pool often exhibit unfavorable properties including transgene copy number loss and epigenetic silencing over the lifespan of the culture, ultimately lowering transgene transcription and corresponding recombinant protein production, which referred to as production instability. Thus, this challenging situation underscores the urgent desire for a new strategy to satisfy ever-growing industrial production requirements.

The lack of specificity of gene integration, which is often susceptible to genetic instability, causes production instability. Alternatively, in recent years, many investigators have shown that the bottleneck arising from traditional randomized cell line development can be overcome through site-specific integration to insert exogenous pieces of DNA into a precise location, which permits their predictable function, allows high levels of transgene expression and makes it possible to generate homogeneous clones with consistent productivity and stability. The transcription and expression activities of genes are influenced by chromatin structural properties and the environment surrounding the genome, and loci that are capable of facilitating high and stable transgene transcription and expression are termed 'hotspots'. Many significant upfront advances have been made to identify potential hotspots, and a number of promising genome loci have been reported, such as the Hprt, Ywhae, Hipp11, Rosa26, and C12orf35 loci. The C12orf35 gene is located on a telomeric region of chromosome 8 in CHO cells. It is widely known that telomeres are usually noncoding, repetitive sequences distributed at chromosome terminals that act as buffers for those coding sequences further behind and thus enable foreign gene expression without interrupting functional genes. Studies have demonstrated that the C12orf35 gene is a potential locus for the integration of foreign genes in mammalian cells and that disruption of C12orf35 gene expression leads to increased productivities and shorter recovery times during selection pressure in CHO cells. Although the C12orf35 gene has been partially researched in cell line development, very few publicly available reports have systematically validated site-specific integration in cell lines concerning the stability of transgene passage, transgene transcription and expression levels.